1. Field of the Invention
The present invention concerns biologically active compounds related to the bryostatin family of compounds, and to methods of preparing and utilizing the same.
2. Introduction
Cancer is a major cause of death in the developed countries, with more than 500,000 human fatalities occurring annually in the United States. Cancers are generally the result of the transformation of normal cells into modified cells that proliferate excessively, leading to the formation of abnormal tissues or cell populations. In many cancers, cell proliferation is accompanied by dissemination (metastasis) of malignant cells to other parts of the body, which spawn new cancerous growths. Cancers can significantly impair normal physiological processes, ultimately leading to patient mortality. Cancers have been observed for many different tissue and cell types, with cancers of the lung, breast, and colorectal system accounting for about half of all cases.
Currently, about one-third of cancer patients can be cured by surgical or radiation techniques. However, these approaches are most effective with cancerous lesions that have not yet metastasized to other regions of the body. Chemotherapeutic techniques currently cure another 17% of cancer patients. Combined chemotherapeutic and non-chemotherapeutic protocols can further enhance prospects for full recovery. Even for incurable cancer conditions, therapeutic treatments can be useful to achieve remission or at least extend patient longevity.
Numerous anticancer compounds have been developed over the past several decades (e.g., Katzung, 1998; Wilson et al., 1991; Hardman et al., 1996). While these compounds comprise many different classes that act by a variety of mechanisms, one general approach has been to block the proliferation of cancerous cells by interfering with cell division. For example, anthracyclines, such as doxorubicin and daunorubicin, have been found to intercalate DNA, blocking DNA and RNA synthesis and causing strand scission by interacting with topoisomerase II. The taxanes, such as Taxol™ and Taxotere™, disrupt mitosis by promoting tubulin polymerization in microtubule assembly. Cis-platin forms interstrand crosslinks in DNA and is effective to kill cells in all stages of the cell cycle. As another example, cyclophosphamide and related alkylating agents contain di-(2-chloroethyl)-amino groups that bind covalently to cellular components such as DNA.
The bryostatins (Formula A) are a family of naturally occurring macrocyclic compounds originally isolated from marine bryozoa. Currently, there are about 20 known natural bryostatins which share three six-membered rings designated A, B and C, and which differ mainly in the nature of their substituents at C7 (ORA) and C20 (RB) (Pettit, 1996).
The bryostatins exhibit potent activity against a broad range of human cancer cell lines and provide significant in vivo life extensions in murine xenograft tumor models (Pettit et al., 1982; Homung et al., 1992; Schuchter et al., 1991; Mohammad et al., 1998). Doses that are effective in vivo are extremely low, with activities demonstrated for concentrations as low as 1 μg/kg (Schuchter et al., 1991). Among additional therapeutic responses, the bryostatins have been found to promote the normal growth of bone marrow progenitor cells (Scheid, 1994; Kraft, 1996), provide cellular protection against normally lethal doses of ionizing radiation (Szallasi, 1996), and stimulate immune system responses that result in the production of T cells, tumor necrosis factors, interleukins and interferons (Kraft, 1996; Lind, 1993). Bryostatins are also effective in inducing transformation of chronic lymphocytic leukemia cells to a hairy cell type (Alkatib, 1993), increasing the expression of p53 while decreasing the expression of bcl-2 in inducing apoptosis in cancer cells (Maki, 1995; Mohammad, 1995) or at least pre-disposing a cell towards apoptosis, and reversing multidrug resistance (MDR) (Spitaler, 1998).
At the molecular level, bryostatins have been shown to competitively inhibit the binding of plant-derived phorbol esters and endogenous diacyl glycerols to protein kinase C (PKC) at nanomolar to picomolar drug concentrations (DeVries, 1998), and to stimulate comparable kinase activity (Kraft, 1986; Berkow, 1985; Ramsdell, 1986). Unlike the phorbol esters, however, the bryostatins do not act as tumor promoters. Thus, the bryostatins appear to operate through a mode of action different from, and complementary to, the modes of action of established anticancer agents; human clinical trials are presently evaluating bryostatin combination therapy with cisplatin or taxol.
Various studies have demonstrated good affinity for bryostatins in which RA is hydroxyl, acetyl, pivaloyl, or n-butanoate, and RB is H, acetyl, n-butanoate, or 2,4-unsaturated octanoate, as measured by PKC binding assay (Wender et al., 1988). The double bond between C13 and C30 can be hydrogenated or epoxidized without significant loss of binding affinity. Hydrogenation of the C21–C34 alkene or acetylation of the C26 hydroxyl, on the other hand, can significantly reduce binding affinity. Inversion of the stereoconfiguration at C26 leads to modest loss of activity (approx. 30-fold) and the suggestion that the methyl group may limit rotation of bonds proximate to the methyl group and contribute to the apparent high binding affinity observed for the bryostatins. Elimination of the hydroxyl at C19 (with concomitant omission of the C20 RB group) causes an approximately 100-fold to 200-fold decrease in binding. Likewise, impairing the accessibility of the C26 hydroxymethyl moiety by replacement of the C26 hydroxyl, or by replacing the methyl or hydrogen substituents of C26 with a tert-butyl or similar bulky substituent, has been proposed for diminishing toxicity (Blumberg et al., 1997).
Although the bryostatins have been known for some time, their low natural abundance, difficulties in isolation and severely limited availability through total synthesis have impeded efforts to elucidate their mode of action and to advance their clinical development. Recently, synthetic analogues of bryostatin were reported wherein the C4–C14 spacer domain was replaced with simplified spacer segments using a highly efficient esterification-macrotransacetalization (Wender et al., 1998a, 1998b). The reported analogues retained orientation of the C1-, C19-, C26-oxygen recognition domain as determined by NMR spectroscopic comparison with bryostatin and varying degrees of PKC-binding affinity. The one analogue tested for in vitro inhibition in human tumor cell lines was reported to possess significant activity. It has remained, however, desired to provide new, simplified, and more readily accessible synthetic agents based on the bryostatin structure to further elucidate the molecular basis of bryostatin's activity and develop improved and more readily available clinical candidates, especially for anticancer applications.